Regulation of cytoskeletal-membrane associations by "on-off" mechanisms is crucial for all cells. The most clearly demonstrated "on-off" mechanism occurs in the red blood cell (RBC), where the cytoskeleton is attached to transmembrane receptors. These cytoskeletal-membrane associations are involved in assembly and regulation of the cytoskeleton, as well as maintenance of membrane integrity. A polyphosphoinositide (PPI) is an obligate cofactor binding to membrane glycophorin in such a way that protein 4.1, and spectrin-4.1-actin, will then associate with the cytoplasmic segment of glycophorin. This "on-off" mechanism requiring PPI may explain effects of Ca++, ATP, and polyphosphates on the cytoskeleton and cell shape. The objectives of this proposal are to: i) Define the structural-functional aspects (subunit structure, A.A. residues involved) of glycophorin's association with PPI, other phospholipids, and cytoskeletal components; ii) Determine if spectrin-actin or associations within "junction points" are regulated by the glycophorin-PPI-protein 4.1 association; iii) Relate in vitro to in vivo regulation via PPI metabolism by quantitating the assembly of cytoskeletal elements on membranes with modified PPI content; iv) Explore the role of a membrane-bound protein kinase which phosphorylates protein 4.1 and is "on-off" regulated by glycophorin-PPI. Glycophorin dimers or monomers chemically modified at arginine or lysine residues will be used to study PPI binding by the Hummel-Dreyer method, 4.1-glycophorin associations by direct binding using sedimentation or inhibition of membrane binding. Intramembranous peptides of glycophorin will be used to inhibit dimer formation. Peptides are prepared by proteolytic cleavage of glycophorin, or by chemical synthesis and purification by HPLC. Direct binding or inhibition of binding of cytoskeletal proteins will be used to assay functionality of modified glycophorin. The effect of glycophorin-PPI-protein 4.1 on spectrin-actin interactions or associations within "junction points" will be assayed by sedimentation or sucrose density gradient sedimentation. Some hemolytic diseases result in increased Ca++ (sickle cell), enzyme deficient RBCs may have altered PPI metabolism as a result modifying the cytoskeleton. Glycophorin is the receptor of the malarial parasite P. falciparum, which requires metabolically active clearance of the underlying cytoskeleton before invasion. Many cellular functions involve active roles for the PPIs (i.e., second messengers), or require "on-off" regulation of membrane- cytoskeleton associations. Thus, studying the role of the PPIs as membrane cofactors may be quite important to many pathological defects which impact upon membrane function. The RBC is the perfect, perhaps the only, paradigm to study at the molecular level PPI regulation of cytoskeletal-membrane associations.